Aldehyde and glycosidase-treated soft and bone tissue xenografts

ABSTRACT

The invention provides articles of manufacture comprising substantially non-immunogenic soft and bone tissue xenografts for implantation into humans. The invention further provides methods for preparing soft and bone tissue xenografts by removing at least a portion of a soft or bone tissue from a non-human animal to provide a xenograft; washing the xenograft in saline and alcohol; subjecting the xenograft to cellular disruption treatment; exposing the xenograft to an aldehyde in an amount ranging from about 0.01% to about 0.10%; and digesting the xenograft with a glycosidase and optionally following with a capping treatment. The invention also provides an article of manufacture produced by the above-identified method of the invention. The invention further provides a soft or bone tissue xenograft for implantation into a human including a portion of a soft or bone tissue from a non-human animal, wherein the portion has extracellular components and substantially only dead cells. The extracellular components have substantially no surface carbohydrate moieties which are susceptible to glycosidase digestion. The extracellular components also have an aldehyde in an amount ranging from about 0.01% to about 0.10% crosslinking the proteins of the extracellular components. Each of the xenografts of the invention are substantially non-immunogenic and have substantially the same mechanical properties as a corresponding native soft or bone tissue.

RELATED APPLICATIONS

This application is a continuation in part of U.S. Ser. No. 09/036,171,filed Mar. 6, 1998, now U.S. Pat. No. 5,984,858, which is a continuationin part of application U.S. Ser. No. 08/483,256, filed Jun. 7, 1995, nowU.S. Pat. No. 5,865,849.

FIELD OF THE INVENTION

The present invention relates to the field of treatment of defectivehuman knee joints, heart valves, and bone and in particular, toreplacement and repair of defective or damaged human knee joints, heartvalves, and bone using a substantially immunologically compatible softtissue or bone from a non-human animal.

BACKGROUND OF THE INVENTION

Soft Tissue

The term “soft tissue”, as used herein, refers to cartilaginousstructures, such as meniscus and articular cartilage; ligaments, such asanterior cruciate ligaments; tendons; and heart valves.

1. Meniscal Cartilage Soft Tissue

Specifically, the femoral condyles articulate with the surface plateausof the tibia, through the cartilaginous medial and lateral menisci softtissue, and all of these structures are held in place by variousligaments. The medial and lateral menisci are structures comprised ofcells called fibrochondrocytes and an extracellular matrix of collagenand elastic fibers as well as a variety of proteoglycans. Undamagedmenisci provide shock absorption for the knee by ensuring proper forcedistribution, stabilization, and lubrication for the interacting bonesurfaces within the knee joint, which are routinely exposed to repeatedcompression loading during normal activity. Much of the shock absorbingfunction of the medial and lateral menisci is derived from the elasticproperties inherent to cartilage. When menisci are damaged throughinjury, disease, or inflammation, arthritic changes occur in the kneejoint, with consequent loss of function.

Since joint cartilage in adults does not naturally regenerate to asignificant degree once it is destroyed, damaged adult menisci havehistorically been treated by a variety of surgical interventions.Damaged menisci have been removed and replaced with prosthetic devices.An artificial knee joint having a rigid plastic femoral member and ametal tibial member is disclosed in U.S. Pat. No. 4,034,418. A number ofmeniscus prostheses have been devised which employ resilient materialssuch as silicone rubber or natural rubber, as in U.S. Pat. No. 4,344,193and U.S. Pat. No. 4,502,161. Additional deformable, flexible resilientmaterials for a meniscus prosthesis such as collagen, tendon, orfibrocartilage are disclosed in U.S. Pat. No. 5,092,894 and U.S. Pat.No. 5,171,322. A cartilage replacement apparatus constructed ofpolyethylene plastic filled with small ball bearings or gelatinous fluidis described in U.S. Pat. No. 5,358,525. However, the known artificialprostheses have been unsatisfactory for treatment of damaged menisci,since they are deficient in the elastic, and therefore in theshock-absorbing, properties characteristic of natural menisci. Moreover,the known artificial devices have not proven able to withstand theforces inherent to routine knee joint function.

One of the present inventors provided improved prosthetic menisci inseveral of his earlier patents (U.S. Pat. No. 4,880,429; U.S. Pat. No.5,007,934; U.S. Pat. No. 5,116,374; and U.S. Pat. No. 5,158,574). Thesepatents generally disclose prosthetic menisci formulated from dry,porous matrices of processed natural fibers such as reconstitutedcross-linked collagen, which optionally include glycosaminoglycanmolecules. Generally, the source of collagen for these prostheticmenisci has been animal Achilles tendons or skin. The reconstitutionprocess removes non-collagenous materials such as glycoproteins,proteoglycans, lipids, native glycosaminoglycans, and the like, whichmay confer additional elastic properties to the original tissue.

2. Articular Cartilage Soft Tissue

Articular cartilage soft tissue covers the ends of all bones that formarticulating joints in humans and animals. Articular cartilage is madeof fibrochondrocytes and an extracellular matrix of collagen fibers aswell as a variety of proteoglycans. The cartilage acts in the joint as amechanism for force distribution and as a lubricant in the area ofcontact between the bones. Without articular cartilage, stressconcentration and friction would occur to the degree that the jointwould not permit ease of motion. Loss of the articular cartilage usuallyleads to painful arthritis and decreased joint motion.

Damaged adult articular cartilage has historically been treated by avariety of surgical interventions including repair, replacement, or byexcision. With repair or excision, regeneration of tissue may occur,although the tissue is usually temporary and inadequate to withstand thenormal joint forces.

Replacement of articular cartilage usually has been by allografting(Sengupta et al. (1974) J. Bone Suro. 56B(1):167-177; Rodrigo et al.(1978) Clin Orth. 134:342-349) by periosteal grafts (see, e.g., Engkvist(1979) Scan J. Plast. Reconstr. Suro. 13:361-369; Rubak (1982) ActaOrthop. Scan. 53:181-186) or with metal and/or plastic components(Rubash et al., eds. (1991) Clin. Orth. Rel. Res. 271:2-96).Allografting dead cartilage tissue has been tried for years with minimalsuccess. This approach has been only partially successful over the longterm due to the host's immunologic response to the graft, failures inthe cryopreservation process, and failures of the attachment sites.Replacement of an entire joint surface with metal and plastic componentshas met excellent success for the older, more sedentary patients, but isgenerally considered insufficient for tolerating the impact of athleticactivities, and has not been shown to restore normal joint mechanics.

In alternative prior art approaches, articular cartilage has beenreplaced with prostheses composed of bone and/or artificial materials.For example, U.S. Pat. No. 4,627,853 describes the use of demineralizedallogenic or xenogeneic bone segments as replacements. The properfunctioning of these replacements depends on the differentialdemineralization of the bone segments. U.S. Pat. No. 4,846,835 describesa grafting technique for transplantation of fibrochondrocytes to promotehealing lesions in articular cartilage. U.S. Pat. No. 4,642,120describes the use of gel-like compositions containing embryonalfibrochondrocytes. U.S. Pat. No. 5,306,311 describes a prostheticarticular cartilage which includes a dry, porous volume matrix adaptedto have in vivo an outer contour substantially the same as that ofnatural articular cartilage.

Despite these developments, the replacement of articular cartilage softtissue with structures consisting of permanent artificial materialsgenerally has been less than satisfactory, and a structure suitable asarticular cartilage and constructed from natural resorbable materials,or analogs thereof, has not been developed. Because the opposingarticular cartilage of mammalian joints is so fragile, it will notwithstand abrasive interfaces nor compliance variances from normal whicheventually result from the implantation of prior art artificialcartilage. Additionally, joint forces are multiples of body weightwhich, in the case of the knee and hip, are typically encountered over amillion cycles per year. Thus far, prior art permanent artificialcartilages have not been composed of materials having natural articularcartilage properties, nor have they been able to be positioned securelyenough to withstand such routine forces.

3. Ligament Soft Tissue

Anterior cruciate ligament soft tissue of the knee (hereinafter the ACL)functions to resist anterior displacement of the tibia from the femur atall flexion positions. The ACL also resists hyperextension andcontributes to rotational stability of the fully extended knee duringinternal and external tibial rotation. The ACL may play a role inproprioception. The ACL is made up of connective tissue structurescomposed of cells, water, collagen, proteoglycans, fibronectin, elastin,and other glycoproteins. Cyril Frank, M. D. et al., Normal Ligament:Structure, Function, and Composition. Injury and Repair of theMusculoskeletal Soft Tissues, 2:45-101. Structurally, the ACL attachesto a depression in the front of the intercondyloid eminence of the tibiaextending postero-superiorly to the medial wall of the lateral femoralcondyle.

The preferred treatment of damaged ACL is ligament reconstruction, usinga bone-ligament-bone autograft. Cruciate ligament reconstruction has theadvantage of immediate stability and a potential for immediate vigorousrehabilitation. However, the disadvantages to ACL reconstruction aresignificant: for example, normal anatomy is disrupted when the patellartendon or hamstring tendons are used for the reconstruction; placementof intraarticular hardware is required for ligament fixation; andanterior knee pain frequently occurs. Moreover, recent reviews ofcruciate ligament reconstruction indicate an increased risk ofdegenerative arthritis with intraarticular ACL reconstruction in largegroups of patients.

A second method of treating ACL injuries, referred to as “primaryrepair”, involves suturing the torn structure back into place. PrimaryACL repair has the potential advantages of a limited arthroscopicapproach, minimal disruption of normal anatomy, and an out-patientprocedure under a local anesthetic. The potential disadvantage ofprimary cruciate ligament repair is the perception that over the longterm ACL repairs do not provide stability in a sufficient number ofpatients, and that subsequent reconstruction may be required at a laterdate. The success rate of anterior cruciate ligament repair hasgenerally hovered in the 60% to 70% range.

4. Heart Valve Soft Tissue

Heart valves are composed of fibrochondrocytes and an extracellularmatrix of collagen and elastic fibers, as well as a variety ofproteoglycans. Various synthetic and tissue based materials (the lattereither from the recipient organism or from a different organism withinthe same species) have been used for forming heart valve replacements.Each have their advantages and disadvantages.

In the case of synthetic heart valves, it may be possible to modifyadvantageously the properties of the heart valves by altering themonomers and/or the reaction conditions of the synthetic polymers.Synthetic heart valves may be associated with thromboembolism andmechanical failure, however. See U.S. Pat. No. 4,755,593.

Tissue based heart valves may demonstrate superior blood contactingproperties relative to their synthetic counterparts. Tissue based heartvalves also may be associated with inferior in vivo stability, however.See U.S. Pat. No. 4,755,593.

Pericardial xenograft tissue valves have been introduced as alternativesto the synthetic and the tissue based valves described above. SeeIonescu, M. I. et al., Heart Valve Replacement With The Ionescu-ShileyPericardial Xenograft, J. Thorac. Cardiovas. Surg. 73; 31-42 (1977).Such valves may continue to have calcification and durability problems,however. See Morse, D, ed. Guide To Prosthetic Heart Valves,Springer-Verlag, New York, 225-232 (1985).

Accordingly, there is a need for mechanically durable, flexible heartvalves replacements which are capable of contacting the blood and arestable in vivo.

Bone Tissue

Human bone, a hard connective tissue consisting of cells embedded in anextracellular matrix of mineralized ground substance and collagenfibers, (Stedman's Medical Dictionary, Williams & Wilkins, Baltimore,Md. (1995)), is the most frequently transplanted tissue in humans. J. M.Lane et al., Current Approaches to Experimental Bone Grafting, 18Orthopedic Clinics of North America (2) 213 (1987). In the United Statesalone more than 100,000 bone graft or implant procedures are performedevery year to repair or to replace osseous defects resulting fromtrauma, infection, congenital malformation, or malignancy. Id.

Bone grafts and implants are often formed of autologous bone. Id.Transplamtable autologous bone tissue for large defects, particularly inchildren, is often unavailable, however. Id. In addition, autologousbone transplantation may result in postoperative morbidity such as pain,hemorrhage, wound problems, cosmetic disability, infection or nervedamage at the donor site. Id. Further, difficulties in fabricating thedesired functional shape from the transplanted autologous bone tissuemay result in less than optimal filling of the bone defect. Id.

Xenografts

Much of the structure and many of the properties of original soft orbone tissues may be retained in transplants through use of heterograftor xenograft materials, that is, soft or bone tissue from a differentspecies than the graft recipient. For example, tendons or ligaments fromcows or other animals are covered with a synthetic mesh and transplantedinto a heterologous host in U.S. Pat. No. 4,400,833. Flat tissues suchas pig pericardia are also disclosed as being suitable for heterologoustransplantation in U.S. Pat. No. 4,400,833. Bovine peritoneum fabricatedinto a biomaterial suitable for prosthetic heart valves, vasculargrafts, bum and other wound dressings is disclosed in U.S. Pat. No.4,755,593. Bovine, ovine, or porcine blood vessel xenografts aredisclosed in WO 84/03036.

Once implanted in an individual, a xenograft provokes immunogenicreactions such as chronic and hyperacute rejection of the xenograft. Inparticular, bone xenografts may result in increased rates of fracture,resorption and nonunion secondary to immunologic rejection.

The term “chronic rejection”, as used herein refers to an immunologicalreaction in an individual against a xenograft being implanted into theindividual. Typically, chronic rejection is mediated by the interactionof IgG natural antibodies in the serum of the individual receiving thexenograft and carbohydrate moieties expressed on cells, and/or cellularmatrices and/or extracellular components of the xenograft. For example,transplantation of soft tissue cartilage xenografts from nonprimatemammals (e.g., porcine or bovine origin) into humans is primarilyprevented by the interaction between the IgG natural anti-Gal antibodypresent in the serum of humans with the carbohydrate structureGalα1-3Galβ1-4G1cNAc-R (α-galactosyl or α-gal epitope) expressed in thexenograft. K. R. Stone et al., Porcine and bovine cartilage transplantsin cynomolgus monkey: I. A model for chronic xenograft rejection, 63Transplantation 640-645 (1997); U. Galili et al., Porcine and bovinecartilage transplants in cynomolgus monkey: II. Changes in anti-Galresponse during chronic rejection, 63 Transplantation 646-651 (1997). Inchronic rejection, the immune system typically responds within one totwo weeks of implantation of the xenograft.

In contrast with “chronic rejection”, “hyper acute rejection” as usedherein, refers to the immunological reaction in an individual against axenograft being implanted into the individual, where the rejection istypically mediated by the interaction of IgM natural antibodies in theserum of the individual receiving the xenograft and carbohydratemoieties expressed on cells. This interaction activates the complementsystem causing lysis of the vascular bed and stoppage of blood flow inthe receiving individual within minutes to two to three hours.

The term “extracellular components”, as used herein, refers to anyextracellular water, collagen and elastic fibers, proteoglycans,fibronectin, elastin, and other glycoproteins, which are present in softtissue; and to mineralized ground substance, bone and collagen fibers,which are present in bone tissue. Stedman's Medical Dictionary, Williams& Wilkins, Baltimore, Md. (1995).

Xenograft materials may be chemically treated to reduce immunogenicityprior to implantation into a recipient. For example, glutaraldehyde isused to cross-link or “tan” xenograft tissue in order to reduce itsantigenicity, as described in detail in U.S. Pat. No. 4,755,593. Otheragents such as aliphatic and aromatic diamine compounds may provideadditional crosslinking through the side chain carboxyl groups ofaspartic and glutamic acid residues of the collagen polypeptide.Glutaraldehyde and diamine tanning also increases the stability of thexenograft tissue.

Xenograft tissues may also be subjected to various physical treatmentsin preparation for implantation. For example, U.S. Pat. No. 4,755,593discloses subjecting xenograft tissue to mechanical strain by stretchingto produce a thinner and stiffer biomaterial for grafting. Tissue forallograft transplantation is commonly cryopreserved to optimize cellviability during storage, as disclosed, for example, in U.S. Pat. No.5,071,741; U.S. Pat. No. 5,131,850; U.S. Pat. No. 5,160,313; and U.S.Pat. No. 5,171,660. U.S. Pat. No. 5,071,741 discloses that freezingtissues causes mechanical injuries to cells therein because ofextracellular or intracellular ice crystal formation and osmoticdehydration.

SUMMARY OF THE INVENTION

The present invention provides a substantially non-immunogenic soft orbone tissue xenograft for implantation into a human in need of soft orbone tissue repair or replacement. The invention further providesmethods for processing xenogeneic soft or bone tissue with reducedimmunogenicity but with substantially native elasticity and load-bearingcapabilities for xenografting into humans.

As used herein, the term “xenograft” is synonymous with the term“heterograft” and refers to a graft transferred from an animal of onespecies to one of another species. Stedman's Medical Dictionary,Williams & Wilkins, Baltimore, Md. (1995).

As used herein, the term “xenogeneic”, as in, for example, xenogeneicsoft or bone tissue, refers to soft or bone tissue transferred from ananimal of one species to one of another species. Id.

The methods of the invention, include, alone or in combination,treatment with radiation, one or more cycles of freezing and thawing,treatment with a chemical cross-linking agent, treatment with alcohol orozonation. In addition to or in lieu of these methods, the methods ofthe invention include, alone or in combination, in any order, a cellulardisruption treatment, glycosidase digestion of carbohydrate moieties ofthe xenograft, or treatment with proteoglycan-depleting factors (theproteoglycan-depleting factor treatment involving soft tissue xenograftsonly). Optionally, the glycosidase digestion or proteoglycan-depletingfactor treatment can be followed by further treatments, such as, forexample, treatment of carbohydrate moieties of the xenograft withcapping molecules. After one or more of the above-described processingsteps, the methods of the invention provide a xenograft havingsubstantially the same mechanical properties as a native soft or bonetissue.

As used herein, the term “cellular disruption” as in, for example,cellular disruption treatment, refers to a treatment for killing cells.

As used herein, the term “capping molecule(s)”, refers to molecule(s)which link with carbohydrate chains such that the xenograft is no longerrecognized as foreign by the subject's immune system.

As used herein, the terms “to cap” or “capping”, refer to linking acapping molecule such as a carbohydrate unit to the end of acarbohydrate chain, as in, for example, covalently linking acarbohydrate unit to surface carbohydrate moieties on the xenograft.

In one embodiment, the invention provides an article of manufacturecomprising a substantially non-immunogenic soft or bone tissue xenograftfor implantation into a human.

In another embodiment, the invention provides a method of preparing asoft or bone tissue xenograft for implantation into a human, whichincludes removing at least a portion of a soft or bone tissue from anon-human animal to provide a xenograft; washing the xenograft in waterand alcohol; and subjecting the xenograft to at least one treatmentselected from the group consisting of exposure to ultraviolet radiation,immersion in alcohol, ozonation, and freeze/thaw cycling, whereby thexenograft has substantially the same mechanical properties as acorresponding portion of a native soft or bone tissue.

As used herein, the term “portion”, as in, for example, a portion ofsoft tissue, bone tissue, second surface carbohydrate moieties orproteoglycans, refers to all or less than all of the respective softtissue, bone tissue, second surface carbohydrate moieties orproteoglycans of the xenograft.

In another embodiment, the invention provides a method of preparing asoft or bone tissue xenograft for implantation into a human, whichincludes removing at least a portion of a soft or bone tissue from anon-human animal to provide a xenograft; washing the xenograft in waterand alcohol; subjecting the xenograft to a cellular disruptiontreatment; and digesting the xenograft with a glycosidase to removefirst surface carbohydrate moieties, whereby the xenograft hassubstantially the same properties as a corresponding portion of a nativesoft or bone tissue.

As used herein, the term “first surface carbohydrate moiety (moieties)”refers to a terminal α-galactosyl sugar at the non-reducing end of acarbohydrate chain.

In still other embodiments, this method can include additional stepssuch as, for example, treating second surface carbohydrate moieties onthe xenograft with capping molecules to cap at least a portion of thesecond surface carbohydrate moieties, whereby the xenograft issubstantially non-immunogenic.

As used herein, the term “second surface carbohydrate moiety (moieties)”refers to a N-acetyllactosamine residue at the non-reducing end of acarbohydrate chain, the residue being non-capped either naturally or asa result of prior cleavage of an α-galactosyl epitope.

In yet another embodiment, the invention provides a method of preparinga bone xenograft for implantation into a human, which includes removingat least a portion of a bone from a non-human animal to provide axenograft; washing the xenograft in water and alcohol; and subjectingthe xenograft to sterilization and cellular disruption treatments,whereby the xenograft has substantially the same properties as acorresponding portion of a native bone and is substantiallynon-immunogenic.

In a further embodiment, the invention provides a method of preparing asoft tissue xenograft for implantation into a human, which includesremoving at least a portion of soft tissue from a non-human animal toprovide a xenograft; washing the xenograft in water and alcohol;subjecting the xenograft to a cellular disruption treatment; anddigesting the xenograft with a proteoglycan-depleting factor to removeat least a portion of the proteoglycans from the xenograft, whereby thexenograft has substantially the same mechanical properties as acorresponding portion of a native soft tissue and is substantiallynon-immunogenic.

In yet a further embodiment, the invention provides a method ofpreparing a xenograft formed of a soft or bone tissue for implantationinto a human, which comprises removing at least a portion of the soft orbone tissue from a non-human animal to provide a xenograft; washing thexenograft in water and alcohol; subjecting the xenograft to a cellulardisruption treatment; exposing the xenograft to an aldehyde in an amountranging from about 0.01% to about 0.10%; and digesting the xenograftwith a glycosidase to remove substantially first surface carbohydratemoieties from the xenograft, whereby the xenograft is substantiallynon-immunogenic and has substantially the same mechanical properties asthe native soft or bone tissue.

In still yet further embodiments, the invention provides articles ofmanufacture including substantially non-immunogenic soft or bone tissuexenografts for implantation into humans produced by one or more of theabove-identified methods of the invention.

In another embodiment, the invention provides a soft or bone tissuexenograft for implantation into a human which includes a portion of asoft or bone tissue from a non-human animal, wherein the portion hassubstantially no surface carbohydrate moieties which are susceptible toglycosidase digestion, and whereby the portion has substantially thesame mechanical properties as a corresponding portion of a native softor bone tissue.

In yet another embodiment, the invention provides a soft or bone tissuexenograft for implantation into a human which includes a portion of asoft or bone tissue from a non-human animal, wherein the portionincludes extracellular components and substantially only dead cells, theextracellular components and dead cells having substantially no surfaceα-galactosyl moieties and having capping molecules linked to at least aportion of surface carbohydrate moieties. The portion of the soft orbone tissue is substantially non-immunogenic and has substantially thesame mechanical properties as the native soft or bone tissue.

In a further embodiment, the invention provides a bone xenograft forimplantation into a human which includes a sterilized, cellulardisrupted portion of a bone from a non-human animal, whereby the portionis substantially non-immunogenic and has substantially the samemechanical properties as a corresponding portion of a native bone.

In still a further embodiment, the invention provides a soft tissuexenograft for implantation into a human which includes a portion of asoft tissue from a non-human animal, wherein the portion includesextracellular components and substantially only dead cells, theextracellular components having reduced proteoglycans. The portion ofthe soft tissue is substantially non-immunogenic and has substantiallythe same mechanical properties as the native soft tissue.

In yet still a further embodiment, the invention provides a xenograftformed of a soft or bone tissue for implantation into a human comprisinga portion of the soft or bone tissue from a nonhuman animal, wherein theportion includes a plurality of extracellular components, a plurality ofsubstantially only dead cells, and an aldehyde in an amount ranging fromabout 0.01% to about 0.10% crosslinking a plurality of proteins of theextracellular components, the extracellular components and the deadcells having substantially no surface carbohydrate moieties which aresusceptible to glycosidase digestion, and whereby the portion issubstantially non-immunogenic and has substantially the same mechanicalproperties as a corresponding portion of the native soft or bone tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The various features of the invention may be more fully understood fromthe following description when read together with the accompanyingdrawings.

FIG. 1 shows a simplified diagrammatic representation of a human kneejoint, with medial and lateral menisci in their native positions.

FIG. 2 is a diagrammatic representation of a cut-away view of a humanknee joint, showing the medial and lateral menisci as they arepositioned in vivo over the medial and lateral condyles of the tibia.

FIG. 3 is a diagrammatic representation of resection of a tom lateralmeniscus of a human knee, and preparation of the knee for receipt of ameniscal implant.

FIG. 4 is a diagrammatic representation the preferred drill guideplacement for posterior lateral meniscal horn insertion into a humanknee.

FIG. 5 is a diagrammatic representation of a cannulated drilloverdrilling guide wire at the posterior lateral meniscal horn insertioninto a human knee.

FIG. 6 is a diagrammatic representation of the appearance of a humanknee with posterior and anterior drill holes for meniscal horninsertion.

FIG. 7 is a diagrammatic representation of the preferred suture passerplacement for pulling a meniscal implant into a human knee joint.

FIG. 8 is a diagrammatic representation of the appearance of a humanknee containing a meniscal implant during the insertion stage.

FIG. 9 is a diagrammatic representation of the appearance of a humanknee containing a meniscal implant with zone-specific meniscal repairsutures in place for final tying of the meniscal repair sutures.

FIG. 10 is shows a simplified diagrammatic representation of a humanknee joint 3, showing the normal positioning of articular cartilage 7 onthe articulating end of femur 2 and articular cartilage 8 on thearticulating end of tibia 4.

FIG. 11 is a graphical representation of the specificity of monoclonalanti-Gal antibodies for α-galactosyl epitopes on bovine serum albumin(BSA), bovine thyroglobulin, mouse laminin, Galβ1-4 G1cNAc-BSA(N-acetyllactosamine-BSA), Galα1-4Galβ1-4G1cNAc-BSA (P1 antigen linkedto BSA), and human thyroglobulin or human laminin.

FIG. 12 is a graphical representation of α-galactosyl epitopeelimination from α-galactosidase treated meniscal cartilage.

FIG. 13 shows a portion of a bone having a defect; and

FIG. 14 shows the bone portion of FIG. 13 with a xenograft of theinvention in the defect.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed against the chronic rejection ofxenografts for implantation into humans. Accordingly, the soft or bonetissue xenograft produced in accordance with the method of the inventionis substantially non-immunogenic, while generally maintaining themechanical properties of a native soft or bone tissue.

While the soft or bone tissue may undergo some shrinkage duringprocessing, a soft tissue xenograft prepared in accordance with theinvention will have the general appearance of a native soft or bonetissue xenograft. For example, a medial meniscus xenograft prepared inaccordance with the invention will have the general appearance of anative medial meniscus, and a lateral meniscus xenograft of theinvention will have the general appearance of a native lateral meniscus.The soft or bone tissue xenograft may also be cut into segments, each ofwhich may be implanted into a recipient as set forth below.

The invention provides, in one embodiment, a method for preparing orprocessing a xenogeneic soft or bone tissue for engraftment into humans.The soft or bone tissue may be harvested from any non-human animal toprepare the xenografts of the invention. Soft or bone tissue fromtransgenic non-human animals or from genetically altered non-humananimals may also be used as xenografts in accordance with the presentinvention. Preferably, bovine, ovine, or porcine knee joints, and morepreferably porcine knee joints, serve as sources of the medial andlateral menisci and articular cartilage soft tissue used to prepare thexenografts. Preferably, bovine and porcine joints, and more preferablyporcine joints, serve as the source of the ligament soft tissuexenografts. Preferably, porcine peritoneum serves as the source of thesoft tissue used to prepare the heart valve xenografts. Alternatively,porcine pericardium can be used to form the heart valve xenografts ofthe present invention. Preferably, bovine, ovine, or porcine bones, andmore preferably porcine bones, serve as sources of the bone used toprepare the xenografts. More preferably, immature animal joints are thesources of the soft tissue, since the soft tissue of younger animals maybe inherently more elastic and engraftable than that of older animals.More preferably, immature pig, calf or lamb bones are the sources of thebone, since the bone of younger animals consists of more cancellous boneand may be less brittle than that of older animals. Most preferably, theage of the source animal is between six and eighteen months at time ofslaughter. Additionally, the patellar tendon, the anterior or posteriorcruciate ligaments, the Achilles tendon or the hamstring tendons may beharvested from the animal source and used as a donor ligament.

In the first step of the method of the invention, an intact soft tissueis removed from a non-human animal. Medial or lateral meniscus areremoved from the knee joints of the non-human animal. Articularcartilage are removed from any joint of the non-human animal. Ligamentsand tendons, such as, for example, the Achilles tendon, as well asintact bone portions are also removed from non-human animals. Preferablysoft tissue from a corresponding joint is used to make the soft tissuexenograft of the invention. For example, articular cartilage from afemuro-tibial (stifle) joint is used to make an articular cartilagexenograft for implantation into a knee. Similarly, articular cartilagefrom a donor animal's hip joint is used to make an articular cartilagexenograft for a human hip joint.

The source of the soft or bone tissues should be collected from freshlykilled animals and preferably immediately placed in a suitable sterileisotonic or other tissue preserving solution. Harvesting of the soft andbone tissues should occur as soon as possible after slaughter of theanimal and preferably should be performed in the cold, i.e., in theapproximate range of about 5° C. to about 20° C., to minimize enzymaticdegradation of the soft and bone tissues.

The soft and bone tissue portions are harvested in the cold, understrict sterile technique.

With respect to meniscal soft tissue, the joint is opened by firsttransecting the patellar tendon. The horns of the menisci are dissectedfree of adhering tissue. A small amount of bone representing asubstantially cylindrical plug of approximately five millimeters indiameter by five millimeters in depth may be left attached to the horns.The meniscal synovial junction is carefully identified and freed fromthe meniscus tissue itself, thereby forming the xenograft.

With respect to articular cartilage soft tissue, a fine peel ofarticular cartilage with a small layer of subchondral bone is shavedfrom the donor joint to form the xenograft.

With respect to ligament soft tissue, the donor joint is opened bystandard surgical technique. Preferably, the ligament is harvested witha block of bone attached to one or both ends, although in some forms ofthe invention the ligament alone is harvested. In one form of theinvention, a block of bone representing a substantially cylindrical plugof approximately 9-10 mm in diameter by approximately 20-40 mm in lengthmay be left attached to the ligament. The ligament is carefullyidentified and dissected free of adhering tissue, thereby forming thexenograft.

With respect to heart valve soft tissue, porcine peritoneum orpericardium is harvested to form the heart valve xenografts according toprocedures known to those of ordinary skill in the art. See, forexample, the peritoneum harvesting procedure discussed in U.S. Pat. No.4,755,593 by Lauren.

With respect to the bone tissue, the harvested bone portion is cut upinto strips or blocks and provided with and without cancellous boneattached to cortical bone.

The xenograft is then washed in about ten volumes of sterile cold waterto remove residual blood proteins and water soluble materials. Thexenograft is then immersed in alcohol at room temperature for about fiveminutes, to sterilize the tissue and to remove non-collagenousmaterials.

A meniscus soft tissue xenograft appears as a shiny “C”-shaped fibroustissue, having generally a crescent-shaped principal surface on the topside (the “superior surface”) and a generally crescent-shaped principalsurface on the bottom side (the “inferior surface”), where the outerportions of the superior and inferior surfaces are joined by way of anouter lateral surface and the inner portions of the superior andinferior surfaces are joined by way of an inner lateral surface.

The articular cartilage soft tissue xenograft appears as a hyalinetissue supported on a bone substrate, having generally aspherical-shaped principal surface on the tip side (the “superiorsurface”), with the under surface of the bone (the “inferior surface”)being rough.

After alcohol immersion, the xenograft may be directly implanted or maybe subjected to at least one of the following treatments: radiationtreatment, treatment with alcohol, ozonation, one or more cycles offreezing and thawing, and/or treatment with a chemical cross-linkingagent. When more than one of these treatments is applied to thexenograft, the treatments may occur in any order.

In one embodiment of the method of the invention, the xenograft may betreated by exposure to ultraviolet radiation for about fifteen minutesor gamma radiation in an amount of about 0.5 to 3 MegaRad.

In another embodiment, the xenograft may be treated by again beingplaced in an alcohol solution. Any alcohol solution may be used toperform this treatment. Preferably, the xenograft is placed in a 70%solution of isopropanol at room temperature.

In still another embodiment, the xenograft may be subjected toozonation.

In a further embodiment of the method of the invention, the xenograftmay be treated by freeze/thaw cycling. For example, the xenograft may befrozen using any method of freezing, so long as the xenograft iscompletely frozen, i.e., no interior warm spots remain which containunfrozen soft tissue. Preferably, the xenograft is dipped into liquidnitrogen for about five minutes to perform this step of the method. Morepreferably, the xenograft is frozen slowly by placing it in a freezer.In the next step of the freeze/thaw cycling treatment, the xenograft isthawed by immersion in an isotonic saline bath at room temperature(about 25° C.) for about ten minutes. No external heat or radiationsource is used, in order to minimize fiber degradation.

In yet a further embodiment, the xenograft may optionally be exposed toa chemical agent to tan or crosslink the proteins within theextracellular components, to further diminish or reduce the immunogenicdeterminants present in the xenograft. Any tanning or crosslinking agentmay be used for this treatment, and more than one crosslinking step maybe performed or more than one crosslinking agent may be used in order toensure complete crosslinking and thus optimally reduce theimmunogenicity of the xenograft. For example, aldehydes such asglutaraldehyde, formaldehyde, adipic dialdehyde, and the like, may beused to crosslink the extracellular collagen of the xenograft inaccordance with the method of the invention. Other suitable crosslinkingagents include aliphatic and aromatic diamines, carbodiimides,diisocyanates, and the like.

When an aldehyde such as, for example, glutaraldehyde is used as thecrosslinking agent, the xenograft may be placed in a buffered solutioncontaining about 0.001% to about 5.0% glutaraldehyde and preferably,about 0.01 % to about 5.0% glutaraldehyde, and having a pH of about 7.4.More preferably about 0.01% to about 0.10% aldehyde, and most preferablyabout 0.01% to about 0.05% aldehyde is used. Any suitable buffer may beused, such as phosphate buffered saline ortrishydroxymethylaminomethane, and the like, so long as it is possibleto maintain control over the pH of the solution for the duration of thecrosslinking reaction, which may be from one to fourteen days, andpreferably from one to five days, and most preferably from three to fivedays.

Alternatively, the xenograft can be exposed to a crosslinking agent in avapor form, including, but not limited to, a vaporized aldehydecrosslinking agent, such as, for example, vaporized formaldehyde orglutaraldehyde. The vaporized crosslinking agent can have aconcentration and a pH and the xenograft can be exposed to the vaporizedcrosslinking agent for a period of time suitable to permit thecrosslinking reaction to occur. For example, the xenograft can beexposed to vaporized crosslinking agent having a concentration of about0.001% to about 5.0% and preferably, about 0.01% to about 5.0%, and a pHof about 7.4. More preferably, the xenograft is exposed to the aldehydein an amount ranging from about 0.01% to about 0.10%, and mostpreferably to an aldehyde ranging in an amount from about 0.01% to about0.05%. The xenograft is exposed to the aldehyde for a period of timewhich can be from one to fourteen days, and preferably from one to fivedays, and most preferably from three to five days. Exposure to vaporizedcrosslinking agent can result in reduced residual chemicals in thexenograft from the crosslinking agent exposure.

The crosslinking reaction should continue until the immunogenicdeterminants are substantially eliminated from the xenogeneic softtissue, but the reaction should be terminated prior to significantalterations of the mechanical properties of the xenograft. When diaminesare also used as crosslinking agents, the glutaraldehyde crosslinkingshould occur after the diamine crosslinking, so that any unreacteddiamines are capped. After the crosslinking reactions have proceeded tocompletion as described above, the xenograft should be rinsed to removeresidual chemicals, and 0.01-0.10 M glycine, and preferably, 0.01-0.05 Mglycine may be added to cap any unreacted aldehyde groups which remain.

In addition to or in lieu of the above treatments, the xenograft can besubjected to a cellular disruption treatment to kill the xenograft'scells. Optionally, the cellular disruption treatment precedes or followsdigestion of the xenograft with glycosidases to remove first surfacecarbohydrate moieties from the xenograft. With respect to the softtissue xenograft, in addition or in lieu of the glycosidase treatment,either preceding or following the glycosidase treatment, the soft tissuexenograft is treated with proteoglycan-depleting factors. Further, theglycosidase and/or proteoglycan-depleting factor digestion (the latterfor the soft tissue xenografts only) in turn is optionally followed bylinkage with capping molecules such as fucosyl or n-acetyl glucosaminemolecules to cap surface N-acetyllactosamine ends of carbohydrate chainsof the xenograft.

In embodiments of this method of the invention, the xenograft issubjected to a cellular disruption treatment to kill the cells of thesoft or bone tissue. Typically after surface carbohydrate moieties havebeen removed from living cells and the extracellular components, theliving cells reexpress the surface carbohydrate moieties. Reexpressionof antigenic moieties of a xenograft can provoke continued immunogenicrejection of the xenograft. In contrast, dead cells are unable toreexpress surface carbohydrate moieties. Removal of antigenic surfacecarbohydrate moieties from dead cells and the extracellular componentsof a xenograft substantially permanently eliminates antigenic surfacecarbohydrate moieties as a source of immunogenic rejection of thexenograft.

Accordingly, in the above-identified embodiments, the xenograft of thepresent invention is subjected to freeze/thaw cycling as discussed aboveto disrupt, i.e., to kill the cells of the soft or bone tissue.Alternatively, the xenograft of the present invention is treated withgamma radiation having an amount of 0.2 MegaRad up to about 3 MegaRad.Such radiation kills the soft or bone tissue cells and sterilizes thexenograft. Once killed, the soft or bone tissue cells are no longer ableto reexpress antigenic surface carbohydrate moieties such α-gal epitopeswhich are factors in the immunogenic rejection of the transplantedxenografts.

Either before or after the soft or bone tissue cells are killed, inembodiments of the invention, the xenograft is subjected to in vitrodigestion of the xenograft with glycosidases, and specificallygalactosidases, such as α-galactosidase, to enzymatically eliminateantigenic surface carbohydrate moieties. In particular, α-gal epitopesare eliminated by enzymatic treatment with α-galactosidases, as shown inthe following reaction:

The N-acetyllactosamine residues are epitopes that are normallyexpressed on human and mammalian cells and thus are not immunogenic. Thein vitro digestion of the xenograft with glycosidases is accomplished byvarious methods. For example, the xenograft can be soaked or incubatedin a buffer solution containing glycosidase. In addition, the xenograftcan be pierced to increase permeability, as further described below.Alternatively, a buffer solution containing the glycosidase can beforced under pressure into the xenograft via a pulsatile lavage process.

Elimination of the α-gal epitopes from the xenograft diminishes theimmune response against the xenograft. The α-gal epitope is expressed innonprimate mammals and in New World monkeys (monkeys of South America)as 1×10⁶35×10⁶ epitopes per cell, as well as on macromolecules such asproteoglycans of the extracellular components. U. Galili et al., Man,apes, and Old World monkeys differ from other mammals in the expressionof -galactosyl epitopes on nucleated cells, 263 J. Biol. Chem. 17755(1988). This epitope is absent in Old World primates (monkeys of Asiaand Africa and apes) and humans, however. Id. Anti-Gal is produced inhumans and primates as a result of an immune response to α-gal epitopecarbohydrate structures on gastrointestinal bacteria. U. Galili et al.,Interaction between human natural anti-α-galactosyl immunoglobulin G andbacteria of the human flora, 56 Infect. Immun. 1730 (1988); R. M.Hamadeh et al., Human natural anti-Gal IgG regulates alternativecomplement pathway activation on bacterial surfaces, 89 J. Clin. Invest.1223 (1992). Since nonprimate mammals produce α-gal epitopes,xenotransplantation of xenografts from these mammals into primatesresults in rejection because of primate anti-Gal binding to theseepitopes on the xenograft. The binding results in the destruction of thexenograft by complement fixation and by antibody dependent cellcytotoxicity. U. Galili et al., Interaction of the natural anti-Galantibody with α-galactosyl epitopes: A major obstacle forxenotransplantation in humans, 14 Immunology Today 480 (1993); M.Sandrin et al., Anti-pig IgM antibodies in human serum reactpredominantly with Galα1-3Gal epitopes, 90 Proc. Natl. Acad. Sci. USA11391 (1993); H. Good et al., Identification of carbohydrate structureswhich bind human anti-porcine antibodies: implications for discordantgrafting in man. 24 Transplant. Proc. 559 (1992); B. H. Collins et al.,Cardiac xenografts between primate species provide evidence for theimportance of the α-galactosyl determinant in hyperacute rejection, 154J. Immunol. 5500 (1995). Furthermore, xenotransplantation results inmajor activation of the immune system to produce increased amounts ofhigh affinity anti-Gal. Accordingly, the substantial elimination ofα-gal epitopes from cells and from extracellular components of thexenograft, and the prevention of reexpression of cellular α-gal epitopescan diminish the immune response against the xenograft associated withanti-Gal antibody binding with α-gal epitopes.

Further, the cartilage soft tissue xenografts of the present inventionare particularly well suited to in vitro enzymatic elimination of theα-gal epitopes. In contrast to organs and other tissues, the cartilageextracellular components undergo extremely slow turnover. Moreover, oncethe cartilage cells, i.e., the fibrochondrocytes are killed, these cellsare prevented from reexpressing the α-gal epitopes, as discussed above.

In addition, the soft or bone tissue xenografts may be treated withpolyethylene glycol (PEG) prior to or concurrently with treatment withglycosidase. PEG acts as a carrier for the glycosidase by covalentlybonding to the enzyme and to the collagen extracellular components.Further, PEG-treated xenografts have reduced immunogenicity.

With respect to the soft tissue xenografts, either before or after suchtissue cells are killed, in embodiments of the invention, the xenograftis washed or digested with one or more different types ofproteoglycan-depleting factors. The proteoglycan-depleting factortreatment can precede or follow glycosidase treatment. Proteoglycanssuch as glycosaminoglycans (GAGs) are interspersed either uniformly asindividual molecules or within varying amounts within the extracellularcomponents of the present invention's xenograft. The GAGs includemucopolysaccharide molecules such as chondroitin 4-sulfate, chondroitin6-sulfate, keratan sulfate, dermatan sulfate, heparin sulfate,hyaluronic acid, and mixtures thereof. The proteoglycans including suchGAGs contain attached carbohydrates such as α-gal epitopes. Suchepitopes stimulate an immune response once the xenograft istransplanted, as discussed above. Washing or digesting the xenograftwith the proteoglycan-depleting factor removes at least a portion of theproteoglycans and attached α-gal epitopes from the extracellularcomponents of the xenograft, and thereby diminishes the immune responseagainst the xenograft upon its transplantation. After theproteoglycan-depleting factor treatment and subsequent transplantation,natural tissue can repopulate the remaining collagen shell.

Non-limiting examples of the proteoglycan-depleting factors used in thepresent invention include proteoglycan-depleting enzymes such aschondroitinase ABC, hyaluronidase, chondroitin AC II lyase, keratanase,and trypsin.

Other proteoglycan-depleting factors used in the present inventioninclude fragments of fibronectin. Homanberg et al. suggest thatfibronectin fragments, such as the amino-terminal 29-kDa fragment, bindto the superficial surface of articular cartilage soft tissue andpenetrate the cartilage to surround the cartilage cells. G. A.Homandberg et al., Fibronectin-fragment-induced cartilage chondrolysisis associated with release of catabolic cytokines, Biochem. J. (1997)321, 751-757; G. A. Homandberg et al., Hyaluronic acid suppressesfibronectin fragment mediated cartilage chondrolysis: I. In vitro,Osteoarthritis and Cartilage (1997) 5, 309-319; G. A. Homandberg et al.,High concentrations of fibronectin fragments cause short-term cataboliceffects in cartilage tissue while lower concentrations cause continuousanabolic effects, Archives Of Biochemistry And Biophysics, Vol. 311, No.2, June, pp. 213-218 (1994); G. A. Homandberg et al., Agents that blockfibronectin fragment-mediated cartilage damage also promote repair,Inflammation Research 46 (1997) 467-471. At selected concentrations,Homanberg et al. further suggest that the addition of such fibronectinfragments to cartilage in vitro or in vivo results in the temporarysuppression of proteoglycan synthesis and the enhancement ofextracellular metalloproteinases which in turn cause a rapidproteoglycan loss from cartilage tissue. Id.

Other proteoglycan-depleting factors known to those of ordinary skill inthe art are also possible for use with the present invention, however.The present invention's xenograft is treated with proteoglycan-depletingfactor in an amount effective for removing at least a portion of theproteoglycans from the extracellular components of the xenograft.Preferably, the xenograft is treated with proteoglycan-depleting factorsuch as hyaluronidase in an amount ranging from about 1.0 TRU/ml toabout 100.0 TRU/ml or proteoglycan-depleting factor such aschondroitinase ABC in an amount ranging from about 0.01 u/ml to about2.0 u/ml or most preferably, in an amount ranging from about 1.0 ul/mlto about 2.0 μ/ml. The xenograft can also be treated withproteoglycan-depleting factor such as fibronectin fragment, (e.g., aminoterminal 29-kDa fibronectin fragment) in an amount ranging from about0.01 μM to about 1.0 μM, and preferably in an amount ranging from about0.1 μM to about 1.0 μM.

Following treatment with glycosidase or treatment withproteoglycan-depleting factors (the latter for soft tissue xenograftsonly), the remaining carbohydrate chains (e.g., glycosaminoglycans) ofthe xenograft are optionally treated with capping molecules to cap atleast a portion of the remaining carbohydrate chains. Treatment withcapping molecules is applicable to both glycosidase-treated andnon-glycosidase-treated xenografts, however. For example, xenograftsfrom knock out animals which may lack α-gal epitopes may be treated withcapping molecules to cap carbohydrate moieties on the xenograft, therebyreducing the xenograft's immunogenicity. Examples of capping moleculesused in the present invention include fucosyl and N-acetyl glucosamine.

Prior to treatment, the outer surface of the xenograft (e.g., the outerlateral surface of meniscus soft tissue xenografts) optionally may bepierced to increase permeability to agents used to render the xenograftsubstantially non-immunogenic. A sterile surgical needle such as an 18gauge needle may be used to perform this piercing step, or,alternatively a comb-like apparatus containing a plurality of needlesmay be used. The piercing may be performed with various patterns, andwith various pierce-to-pierce spacings, in order to establish a desiredaccess to the interior of the xenograft. Piercing may also be performedwith a laser. In one form of the invention, one or more straight linesof punctures about three millimeters apart are establishedcircumferentially in the outer lateral surface of the xenograft.

Prior to implantation, the soft or bone tissue xenograft of theinvention may be treated with limited digestion by proteolytic enzymessuch as ficin or trypsin to increase tissue flexibility, or coated withanticalcification agents, antithrombotic coatings, antibiotics, growthfactors, or other drugs which may enhance the incorporation of thexenograft into the recipient joint. The soft tissue xenograft of theinvention may be further sterilized using known methods, for example,with additional glutaraldehyde or formaldehyde treatment, ethylene oxidesterilization, propylene oxide sterilization, or the like. The xenograftmay be stored frozen until required for use.

Further, the bone xenograft of the invention can be treated with anosteoinductive factor in an effective amount to stimulate the conversionof soft tissue cells to osseous tissue formers. Alternatively oradditionally, the osteoinductive factor can be administered directly tothe target defect. As used herein, the term “osteoinductive factor”refers to a protein which stimulates the differentiation of uncommittedconnective tissue cells into bone-forming cells. J. M. Lane et al.,Current Approaches to Experimental Bone Grafting, 18 Orthopedic ClinicsofNorth America (2) 214 (1987).

Methods of preparing and administering the osteoinductive factor to agraft or to the target defect are known in the prior art such as, forexample, Sharon Stevenson, D. V. M., Ph.D., et al., The Effect ofOsteogenic (A Bone Morphogenetic Protein) on the Formation of Bone inOrthotopic Segmental Defects in Rats, 76, A. The Journal of Bone AndJoint Surgery No. 11, 1676-1687 (1994) and John E. Feighan, et al.,Induction of Bone by a Demineralized Bone Matrix Gel: A Study in a RatFemoral Defect Model, 13 Journal of Orthopaedic Research 881-889 (1995).For example, osteoinductive factor in the form of a gel, in the presenceof a synthetic carrier, such as a hydroxyapatite ceramic cylinder, orusing a carrier, such as polyethylene glycol (PEG) or glycerol, or abuffer, such as phosphate, and/or any combination of the above can beadministered to the defect site and/or used to impregnate the xenograft.

The osteoinductive factor is added to the interstices of the xenograftand/or to the defect site in an amount effective to induce boneformation. For example, doses of about 10 mg to about 200 mg ofosteoinductive factor can be added. Examples of osteoinductive factorswhich can be used in the present invention include bone morphogenicproteins (BMP) and associated non-collagenous proteins (NCP). Suchosteoinductive factors are commercially available from, for example,Creative Biomolecules, Inc., Hopkington, Mass.

The bone xenograft of the invention also can be treated with ademineralization agent in an effective amount to remove substantiallyminerals such as, for example, calcium from the extracellular matrix ofthe xenograft. For example, the xenograft of the invention can be soakedin a solution containing demineralization agents, such as, hydrochloricacid, and other demineralization agents known to those of ordinary skillin the art, at a predetermined concentration to demineralizesubstantially the xenograft of the invention. Once the minerals areremoved from the xenograft, a porous volume matrix is formed with poresranging in size from about 50 microns to about 500 microns. It istheorized that the collagen of demineralized extracellular bone matrixserves as an osteoconductive scaffolding and facilitates the migrationof bone forming components once bone graft is implanted. J. M. Lane etal., Current Approaches to Experimental Bone Grafting, 18 OrthopedicClinics ofNorth America (2) 220 (1987). It is further theorized thatdemineralized bone possesses greater osteoinductive activity than, forexample, autologous bone, because bone mineral impedes the release ofosteoinductive proteins from extracellular bone matrix. Id. at 218.According to this theory, demineralization enlarges the access ofsurrounding responsive cells to osteoinductive proteins and augments thepotential of the osteoinductive proteins. Such demineralization agentsare commercially available from, for example, Sigma, Inc., St. Louis,Mo.

In addition, a binding agent can be added into the bone xenograft of thepresent invention. As used herein, a binding agent is an adhesionmolecule, or adhesive portion or analog thereof, which aids in boneformation by providing a tacky surface to which bone forming cells canstick. The binding agent is added in an effective amount to facilitatethe attachment of mesenchymal and other differentiated bone formingcells to the extracellular matrix of the bone xenograft. Examples ofbinding agents useful in the present invention include bone cement;fibrin glue; mussel glue, such as mussel glue containing bioadhesivepolyphenolic proteins derived from several species of the mussel genusMytilus (see e.g., U.S. Pat. No. 4,585,585); chondronectin; osteonectin;and fibronectin and arginine-glycine-aspartic acid (RGD) peptide (seee.g., U.S. Pat. No. 5,681,353), a portion of which can be conjugated to,for example, chondroitin sulfate, and other binding agents known tothose persons of ordinary skill in the art. Such binding agents arecommercially available from, for example, Telios Pharmaceuticals, Inc.,San Diego, Calif.

The soft or bone tissue xenograft of the invention, or a segmentthereof, may be implanted into damaged human joints by those of skill inthe art using known arthroscopic surgical techniques. Specificinstruments for performing arthroscopic techniques are known to those ofskill in the art, which ensure accurate and reproducible placement ofsoft tissue implants.

Meniscus Cartilage Soft Tissue Xenograft Implantation

For meniscal cartilage replacement to succeed, the following goals arepreferably accomplished:

1. The torn fragmented pieces of native meniscal cartilage must beremoved.

2. The attachment sites for the meniscal horns must be anatomicallyplaced.

3. The periphery of the meniscal implant must be attached securelyenough to permit axial and rotational loads.

4. The surrounding capsule and ligaments of the knee joint must beneither excessively violated nor constrained by the fixation technique.The method of meniscal implantation described in detail below is derivedfrom K. R. Stone, et al., Arthroscopy: The Journal ofArthroscopic andRelated Surgery 9, 234-237 (1993); other methods of meniscalimplantation may also be employed to use the xenogeneic meniscalxenografts of the present invention.

Initially, complete diagnostic arthroscopy of the knee joint isaccomplished using known methods. If ACL surgery is to be performedsimultaneously, the femural and tibial tunnels for the cruciatereconstruction should be drilled first. The torn portion of the meniscalcartilage is evaluated. If meniscal repair cannot be accomplished due toseverity of the tear or poor quality of the tissue, then preparation ofthe meniscal rim is undertaken by removing the torn portions of thecartilaginous tissue (FIG. 3). When the entire human meniscus is to bereplaced by a xenogeneic meniscus xenograft of the invention, nearly allof the human meniscus is removed. Additionally, for replacement of theentire human meniscus with a xenogeneic meniscus xenograft of theinvention, resection of the human meniscal horns and preparation of bonytunnels to accept bone plugs may be required. When only a portion of thehuman meniscus is to be replaced with a segment of the xenogeneicmeniscus xenograft of the invention, only the damaged portions areremoved, preserving the peripheral rim and horns for attachment of thexenogeneic meniscus xenograft segment. If absolutely no human meniscalrim is present, then partial replacement of the meniscus should not beperformed. If the joint is excessively tight, a joint distractor may beapplied or the medial collateral ligament may be partially released.

For medial or lateral meniscal replacement, the arthroscope is placed inthe mid-lateral or anterior lateral portal and the tibial guide isplaced through the anterior medial portal. The tip of the guide isbrought first to the posterior horn of the meniscus. It should be notedthat the posteromedial horn inserts on the posterior slope of the tibialeminence. A (frill pin is then brought from the anterior medial side ofthe tibial tuberosity to the posterior horn insertion (FIG. 4). The pinplacement can be confirmed by passing the arthroscope through theintercondylar notch and viewing the exit site of the pin. Extreme caremust be undertaken to avoid penetration through the posterior capsule ofthe knee, endangering the neurovascular bundle. When the pin position isconfirmed, the pin is then overdrilled with a 4.5-mm cannulated drillbit with the option of a drill stop to prevent posterior capsularpenetration (FIG. 5). The bit is left in place and used as a tunnel forpassage of a suture passer with a suture such as a #2 EthibondTM sutureavailable from Johnson & Johnson. The suture is passed up the bore ofthe drill bit, the drill bit removed, and the suture left in place.

The anterior medial meniscus insertion point in humans variesconsiderably, most often being found anterior to the medial tibialeminence. The anterior horn of the lateral meniscus inserts justposterior to the anterior cruciate ligament. An anterior drill hole ismade by first identifying the insertion point of the anterior horn ofthe lateral meniscus, by placing the tip of the drill guide so that arelatively vertical hole will be made (FIG. 6). The drill pin is placed,then the cannulated drill bit is used to overdrill the drill pinplacement to form the anterior drill hole. A suture passer is placed inthe anterior drill hole. Alternatively, the anterior horn of the medialmeniscus is affixed with a suture anchor directly to bone as opposed toa drill hole.

Before the suture is grasped from the anterior and posterior drillholes, the anterior portal is widened to approximately 2 cm. The suturegrasper is then passed through the widened portal, and both the anteriorand the posterior sutures brought out simultaneously. This techniqueprevents the sutures from becoming entangled in two different planes ofthe fat pad and capsular tissue.

The implant is now brought onto the field. Two horizontal mattresssutures, for example, #2-0 EthibondT sutures or the like, are placedthrough each horn of the xenogeneic meniscus xenograft with the freeends exiting the inferior surface (FIG. 7). The two posterior suturesare then drawn through the knee and out the posterior tibial tunnel(FIG. 8). If viewing from a mid-lateral portal, the anterolateral portalcan be used for probe insertion to push the implant medially into placethrough a 1-inch incision. No insertion cannula is required. Theanterior sutures are then similarly passed. The horn sutures are thentied over the anterior tibial bony bridge.

Next, zone specific meniscal repair cannulae are brought into place. Formedial insertions, a posterior medial vertical incision is made onethird of the distance from the back of the knee for protection of thesaphenous nerve and for retrieval of the inside-out meniscal repairneedles. A second vertical incision is usually required furtheranteriorly, next to the anterior medial arthroscopy portal, to capturethe anterior exiting needles. Through these two incisions, the sutureneedles can be captured and the knots placed directly over the capsule(FIG. 9).

When using the meniscal repair needles, the posterior cannulae should beused first, with the sutures placed vertically and evenly spaced. Therepair should proceed from posterior to anterior so that a buckle is notproduced within the xenograft. Each knot is tied as it is placed toavoid the chance of suture tangling. The knots are spaced approximately4 mm apart. The knee is cycled through several complete ranges of motionof ensure that the xenograft moves smoothly without impingement.

When performing a lateral meniscal replacement, the medial portal issuitable for xenograft insertion. This may require excision of theligamentous mucosa and removal of a portion of the fat pad. The drillguide for the posterior horn of the lateral meniscus is inserted throughthe anteromedial portal. The posterior slope of the lateral tibial spinemust be identified for accurate meniscal horn insertion. The anteriorhorn inserts on the anterior slope of the lateral tibial spine inapproximation to the lateral aspect of the anterior cruciate ligament.The advantage of drilling these holes from the medial side is that thetunnel divergence will be greater, providing a larger bony bridgebetween the horn insertions. The remainder of the insertion techniqueremains the same, except that great care should be taken to protect theneurovascular bundle when suturing the posterior horn. Accessingposterolateral exposure is necessary to safeguard the common peronealnerve and to expose the lateral capsule. If there is any doubt about thesuture placement, open posterior horn suturing should be performed inthe standard fashion. Alternatively, meniscus and/or stabilizationdevices such as arrows or staples can be used instead of sutures.Stabilization arrows manufactured by Bionix, Inc., Malvern, Pa., arenon-limiting examples of such stabilization arrows. Other stabilizationdevices known to those of ordinary skill in the art can also be used.

Routine skin closure and dressings are applied. Thirty milliliters of0.5% Marcaine (Astra) with epinephrine may be instilled if desired. Apostoperative hinged knee brace may be applied with the range of motionlimited to 30° of extension and 90° of flexion.

Articular Cartilage Soft Tissue Xenograft Implantation

The underlying bone bed of the recipient joint is prepared with a boneburr to produce a cancellous bleeding bed. Grafting can involve eitherthe entire articular surface or a portion of the articular surface. Thesubstantially non-immunogenic articular cartilage xenograft of theinvention is applied to the recipient joint as a cover, which is held inplace by one or more suture anchors, absorbable pins, screws, staples,and the like. A fibrin clot may also be used to hold the substantiallynon-immunogenic articular cartilage xenograft in place.

Ligament Soft Tissue Xenograft Implantation

The irreparably damaged ligament is removed with a surgical shaver. Theanatomic insertion sites for the ligament are identified and drilled toaccommodate a bone plug. The size of the bone plug can be about 9-10 mmin width by about 9-10 mm in depth by about 20-40 mm in length. Thexenogeneic ligament is brought through the drill holes and affixed withinterference screws. Routine closure is performed.

This invention is further illustrated by the following Examples whichshould not be construed as limiting. The contents of all references andpublished patents and patent applications cited throughout theapplication are hereby incorporated by reference.

Heart Valve Soft Tissue Xenograft Implantation

The heart valve xenograft of the invention, or a segment thereof, may beformed of porcine peritoneum or pericardium and may be implanted toreplace and/or to repair damaged heart valves by those of skill in theart using known techniques. Such techniques are performed with specificinstruments which insure accurate and reproducible placement of theimplants and which are known to those of ordinary skill in the art.

Bone Tissue Xenograft Implantation

The bone xenograft of the invention, or a segment thereof, may beimplanted into damaged human joints by those of skill in the art usingknown arthroscopic surgical techniques. Holes in bones are manuallypacked with bone according to standard surgical techniques. Specificinstruments for performing surgical techniques, which ensure accurateand reproducible placement of bone implants are known to those of skillin the art.

EXAMPLE 1

Assay For α-Gal Epitopes' Elimination From Soft Tissue Byα-Galactosidase

In this example, an ELISA assay for assessing the elimination of α-galepitopes from soft tissue is conducted.

A monoclonal anti-Gal antibody (designated M86) which is highly specificfor α-gal epitopes on glycoproteins is produced by fusion of splenocytesfrom anti-Gal producing knock-out mice for α 1,3 galactosyltransferase,and a mouse hybridoma fusion partner.

The specificity of M86 for α-gal epitopes on glycoproteins isillustrated in FIG. 11. M86 binds to synthetic α-gal epitopes linked to-bovine serum albumin (BSA), to ▴-bovine thyroglobulin which has 11α-gal epitopes, R. G. Spiro et al., Occurrence of α-D-galactosylresidues in the thyroglobulin from several species. Localization in thesaccharide chains of complex carbohydrates, 259 J. Biol. Chem. 9858(1984); or to ▪-mouse laminin which has 50 α-gal epitopes, R. G.Arumugham et al., Structure of the asparagine-linked sugar chains oflaminin. 883 Biochem. Biophys. Acta 112 (1986); but not to □-humanthyroglobulin or human laminin, O-Galβ-4 G1cNAc-BSA(N-acetyllactosamine-BSA) and Galα1-4Galβ1-4G1cNAc-BSA (P1 antigenlinked to BSA), all of which completely lack α-gal epitopes. Binding ismeasured at different dilutions of the M86 tissue culture medium.

Once the M86 antibody is isolated, the monoclonal antibody is dilutedfrom about 1:20 to about 1:160, and preferably diluted from about 1:50to about 1:130. The antibody is incubated for a predetermined period oftime ranging between about 5 hr to about 24 hr, at a predeterminedtemperature ranging from about 3° C. to about 8° C. The antibody ismaintained in constant rotation with fragments of soft tissue about 5 μmto about 100 μm in size, and more preferably with soft tissue fragmentsranging from about 10 μm to about 50 μm in size, at various soft tissueconcentrations ranging from about 200 mg/ml to about 1.5 mg/ml.Subsequently, the soft tissue fragments are removed by centrifugation atcentrifugation rate ranging from about 20,000×g to about 50,000×g. Theproportion of M86 bound to the soft tissue is assessed by measuring theremaining M86 activity in the supernatant, in ELISA with α-gal-BSA asdescribed in the prior art in, for example, U. Galili et al., Porcineand bovine cartilage transplants in cynomolgus monkey: II. Changes inanti-Gal response during chronic rejection, 63 Transplantation 645-651(1997). The extent of binding of M86 to the soft tissue is defined as apercentage inhibition of subsequent binding to α-gal-BSA. There is adirect relationship between the amount of α-gal epitopes in the softtissue and the proportion of M86 complexed with the soft tissuefragments, thus removed from the supernatant (i.e., percentageinhibition).

An example of the assay is shown in FIG. 12. Fragments of homogenizedmeniscus cartilage (∘) or meniscus cartilage () treated withα-galactosidase are incubated with the M86 monoclonal antibody (diluted1:100) for 20 hr at 4° C. Subsequently, the meniscus cartilage fragmentsare removed by centrifugation at 35,000×g and the remaining M86 in thesupernatant is assessed in ELISA with α-gal-BSA as solid phase antigen.FIG. 12 shows that treatment of the meniscus cartilage with 200 U/mlα-galactosidase for 4 hour at 30° C. followed by five washes withphosphate-buffered solution (PBS) completely eliminates the α-galepitopes. Thus, since there is no inhibition of subsequent M86 bindingto α-gal-BSA even at a high meniscus cartilage fragment concentration of200 mg/ml.

EXAMPLE 2

Assessment Of Primate Response To Implanted Porcine Meniscus Cartilage,Articular Cartilage, Ligament And Heart Valve Soft Tissue XenograftsTreated With α-Galactosidase

In this example, porcine meniscus cartilage, articular cartilage,ligament, and peritoneum heart valve soft tissue implants are treatedwith α-galactosidase to eliminate α-galactosyl epitopes, the implantsare transplanted into cynomolgus monkeys, and the primate response tothe soft tissue implants is assessed.

Porcine stifle joints are sterilely prepared and meniscus cartilage andarticular cartilage and other surrounding attached soft tissuessurgically removed. Porcine peritoneum is also harvested for formingheat valve xenografts and adherent fatty and/or muscular tissuessurgically removed. The meniscus cartilage, articular cartilage andheart valve soft tissue specimens are washed for at least five minuteswith an alcohol, such as ethanol or isopropanol, to remove synovialfluid and lipid soluble contaminants. The meniscus cartilage, articularcartilage and heart valve soft tissue specimens are frozen at atemperature ranging from about −35° C. to about −90° C., and preferablyat a temperature up to about −70° C., to disrupt, that, is to kill, thespecimens' fibrochondrocytes.

Porcine stifle joints are also sterilely prepared and ligaments, eachwith a block of bone attached to one or both ends, are removed in thecold, under strict sterile technique. Each of the blocks of bonerepresents a substantially cylindrical plug of approximately 9 mm indiameter by about 40 mm in length. Each ligament soft tissue specimen iscarefully identified and dissected free of adhering tissue, therebyforming the xenograft. The ligament soft tissue xenograft specimens arethen washed for at least five minutes with an alcohol, such as ethanolor isopropanol, to remove synovial fluid and lipid soluble contaminants.Subsequently, the specimens are frozen at a temperature of about −70° C.to disrupt, that is, to kill, the ligament specimens' cells.

Each meniscus cartilage, articular cartilage, heart valve and ligamentsoft tissue xenograft specimen is cut into two portions. Each firstportion is immersed in a buffer solution containing α-galactosidase at apredetermined concentration. The specimens are allowed to incubate inthe buffer solutions for a predetermined time period at a predeterminedtemperature. Each second portion is incubated under similar conditionsas the corresponding first portion in a buffer solution in the absenceof α-galactosidase and serves as the control.

At the end of the incubation, the soft tissue xenograft specimens arewashed under conditions which allow the enzyme to diffuse out. Assaysare performed to confirm the complete removal of the α-gal epitopes.

Each meniscus cartilage soft tissue xenograft specimen is implanted inthe supra patellar pouch of six cynomolgus monkeys. With the animalsunder general inhalation anesthesia, an incision of about 1 cm is madedirectly into the supra patellar pouch at the superior medial border ofthe patella extending proximally. A piece of the porcine cartilage softtissue of about 0.5 cm to about 1 cm in length is placed into the pouchwith a single nylon stitch as a marking tag.

The articular cartilage xenograft specimens are implanted in the suprapatellar pouch of six cynomolgus monkeys substantially following theabove-identified implantation procedure.

The porcine peritoneum is formed into heart valves and the heart valvexenograft specimens are implanted in the six cynomolgus monkeysaccording to heart valve implantation procedures known to those ofordinary skill in the art.

The ligament soft tissue xenograft specimens are implanted in sixcynomolgus monkeys using the following implantation procedure. With theanimals under general inhalation anesthesia, the anatomic insertionsites for the xenogeneic ligament are identified and drilled toaccommodate a substantially 9 mm in diameter by 40 mm in length boneplug. The xenogeneic ligament is brought through the drill holes andaffixed with interference screws.

The implantation procedures are performed under sterile surgicaltechnique., and the wounds are closed with 3-0 vicryl or a suitableequivalent known to those of ordinary skill in the art. The animals arepermitted unrestricted cage activity and monitored for any sign ofdiscomfort, swelling, infection, or rejection. Blood samples (e.g., 2ml) are drawn periodically (e.g., every two weeks) for monitoring ofantibodies.

The occurrence of an immune response against the xenograft is assessedby determining anti-Gal and non-anti-Gal anti-soft tissue antibodies(i.e., antibodies binding to soft tissue antigens other than the α-galepitopes) in serum samples from the transplanted monkeys. At least twoml blood samples are drawn from the transplanted monkeys on the day ofimplant surgery and at periodic (e.g., two week) intervalspost-transplantation. The blood samples are centrifuged and the serumsamples are frozen and evaluated for the anti-Gal and other non-anti-Galanti-soft tissue antibody activity.

Anti-Gal activity is determined in the serum samples in ELISA withα-gal-BSA as solid phase antigen, according to methods known in theprior art, such as, for example, the methods described in Galili et al.,Porcine and bovine cartilage transplants in cynomolgus monkey: II.Changes in anti-Gal response during chronic rejection, 63Transplantation 645-651 (1997).

Assays are conducted to determine whether α-galactosidase treatedxenografts induce the formation of anti-soft tissue antibodies. Formeasuring anti-soft tissue antibody activity, ELISA assays are performedaccording to methods known in the prior art, such as, for example, themethods described in K. R. Stone et al., Porcine and bovine cartilagetransplants in cynomolgus monkey: I. A modelfor chronic xenograftrejection, 63 Transplantation 640-645 (1997).

The soft tissue xenograft specimens are optionally explanted at one totwo months post-transplantation, sectioned and stained for histologicalevaluation of inflammatory infiltrates. Post-transplantation changes inanti-Gal and other anti-cartilage soft tissue antibody activities arecorrelated with the inflammatory histologic characteristics (i.e.,granulocytes or mononuclear cell infiltrates) within the explanted softtissue, one to two months post-transplantation, using methods known inthe art, as, for example, the methods described in K. R. Stone et al.,Porcine and bovine cartilage transplants in cynomolgus monkey: I. Amodel for chronic xenograft rejection, 63 Transplantation 640-645(1997).

Where the soft tissue is explanted, the soft tissue xenografts areaseptically harvested, using anesthetic procedure, surgical exposure ofjoints, removal of the implants and closure of the soft tissue (wherethe animals are allowed to recover). At the time of the xenograftremoval, joint fluid, if present in amounts sufficient to aspirate, iscollected from the stifle joints for possible immunologic testing if thegross and histopathologic evaluation of the transplants indicate goodperformance of the transplanted soft tissue.

The animals which have had meniscus cartilage or articular cartilagexenograft implantations are allowed to recover and are monitored closelyuntil the incisions have healed and the gait is normal. The xenograftsamples are collected, processed, and examined microscopically.

Portions of the meniscus cartilage, articular cartilage, heart valve andligament implants and surrounding tissues are frozen in embeddingmediums for frozen tissue specimens in embedding molds forimmunohistochemistry evaluation according to the methods known in theprior art. “TISSUE-TEK®” O.C.T. compound which includes about 10% w/wpolyvinyl alcohol, about 4% w/w polyethylene glycol, and about 86% w/wnonreactive ingredients, and is manufactured by Sakura FinTek, Torrence,Calif., is a non-limiting example of a possible embedding medium for usewith the present invention. Other embedding mediums known to those ofordinary skill in the art may also be used. The remaining implant andsurrounding tissue is collected in 10% neutral buffered fomialin forhistopathologic examination.

EXAMPLE 3

Assessment Of Primate Response To Implanted Meniscus Cartilage,Articular

Cartilage, Ligament And Heart Valve Soft Tissue Xenografts Treated Withα-Galactosidase, Fucosyl and Fucosyltransferase

In this example, porcine meniscus cartilage, articular cartilage,ligament and peritoneum heart valve soft tissue implants are treatedwith α-galactosidase to eliminate α-gal epitopes, as described inExample 2. The soft tissue implants are further treated with fucosyl andfucosyl transferase to cap remaining carbohydrate chains with fucosyl.Fucosyltransferase facilitates the transfer of fucosyl to the xenograft.The fucosyl links to and thus caps the remaining carbohydrate chains.Capping with fucosyl interferes with the ability of the subject's immunesystem to recognize the xenograft as foreign. The soft tissue implantsare transplanted into cynomolgus monkeys, and the primate response tothe soft tissue implants is assessed.

Meniscus cartilage and articular cartilage implants from porcine stiflejoints, heart valve implants from porcine peritoneum and ligamentimplants from porcine stifle joints are prepared as the implants areprepared in Example 2 including the α-galactosidase treatment. Prior toimplantation into the monkeys, however, the implants are further treatedwith a predetermined amount of fucosyl and fucosyltransferase, atspecified concentrations for a predetermined time and at a predeterminedtemperature, to cap remaining carbohydrate chains with fucosyl. Forexample, the implants are immersed in buffer solutions at predeterminedconcentrations of fucosyl and fucosyl transferase. The implants areincubated for a predetermined time period at a predeterminedtemperature.

Other molecules, such as N-acetyl glucosarnine in combination with thecorresponding glycosyltransferase, can also be used for capping thecarbohydrate chains of the implants.

Subsequently, the implants are washed to remove the enzyme and implantedinto the monkeys, and the occurrence of an immune response against thexenograft is assessed as described above in Example 2.

EXAMPLE 4

Assessment Of Primate Response To Implanted Meniscus Cartilage.Articular Cartilage, Ligament And Heart Valve Soft Tissue XenograftsSubjected To Freeze Thaw Cycling And Treatment WithProteoglycan-Depleting Factors.

In this example, porcine meniscus cartilage, articular cartilage,ligament and peritoneum heart valve soft tissue implants are preparedand frozen to disrupt, that is, to kill the specimens' cells, asdescribed above in Example 2. The soft tissue implants are furthertreated with proteoglycan-depleting factors to eliminate substantiallythe proteoglycans from the xenograft. Subsequently, the xenografts aretreated with glycosidase to remove substantially remaining α-galepitopes from the xenograft, as described in Example 2. Substantialelimination of the proteoglycans and the remaining α-gal epitopesinterferes with the ability of the recipient subject's immune system torecognize the xenograft as foreign. The soft tissue implants aretransplanted into cynomologous monkeys, and the primate response to thesoft tissue implants is assessed.

Meniscus cartilage, articular cartilage and ligament implants fromporcine stifle joints and heart valve implants from porcine peritoneumare prepared following the preparation procedures outlined in Example 2including the sterilization, and freeze/thaw cycling treatments. Achondroitinase ABC solution is then prepared by combining 0.05M Tris-HCL(7.88 gm/liter-MW=157.60), 5mM benzamidine-HCL(0.783gm/liter-MW=156.61), 0.010 M N-ethylmaleimide (1.2513gm/liter-MW=125.13), and 0.001M phenylmethylsulfonyl fluoride (0.17420gm/liter-MW=174.2), dissolved in methanol. A mixture of 15 M NaCl (8.775gm/liter-MW=58.5), penicillin and streptomycin (1% (v/v) 1Oml/liter)along with enzyme in the amount of 1 unit chondroitinase ABC (Sigma#C-3509) Enzyme Solution per 1 ml of solution is added to bring thesolution to 1 liter.

Each soft tissue xenograft specimen is incubated in the chondroitinaseABC enzyme solution at a concentration of 1 ml of solution per a 3 mmdiameter soft tissue plug. The incubations are performed at a pH of 8.0and 37 degrees C. in a shaker water bath for 48 hours. After theincubation, each soft tissue specimen is washed in appropriate bufferand the washings are added to the chondroitinase ABC solution. Each softtissue specimen is then re-incubated with the chondroitinase ABCsolution at a concentration of 1 unit chondroitinase ABC (Sigma #C-3509)Enzyme Solution per 1 ml of solution for another 48 hours as describedabove. Each soft tissue specimen is again washed in appropriate buffersolution, and the washings are added to the chondroitinase ABC solution.

Each soft tissue specimen is then incubated in 1 ml of trypsin solution(1 mg/ml trypsin, 0.15 M NaCl, 0.05 M Na Phosphate) at a pH of 7.2 for24 hours. The incubation is performed in a shaker water bath at 37degrees C. Each soft tissue specimen is washed in appropriate buffersolution, and the washings are added to the trypsin solution.

Each specimen is then placed in 1 ml of hyaluronidase solution (0.01mg/mil testicular hyaluronidase, 0.005 M Benzamidine HCL, 001 M PMSF,0.010M Nethylmaleimide, 0.005 M Benzamidine HCL, 1% v/v penicillin andstreptomycin) at a pH 6.0 for 24 hours. The incubation is performed in ashaker water bath at 37 degrees C. Each soft tissue specimen is thenrinsed again in an appropriate buffer solution, and the washings areadded to the hyaluronidase solution.

Subsequently, the implants are treated with glycosidase as describedabove in Example 2, implanted into the monkeys, and the occurrence of animmune response against each of the xenografts is assessed as describedabove in Example 2.

EXAMPLE 5

Assessment Of Response In Mice To Implanted Bone Xenografts Treated Withα-Galactosidase And Demineralized Bone Matrix Gel ContainingOsteoinductive Factor

In this example, porcine bone implants are treated with α-galactosidaseto eliminate α-galactosyl epitopes and impregnated with demineralizedbone matrix gel containing osteoinductive factor to stimulate theconversion of soft tissue formers in the target defect to osseous tissueformers. The implants are transplanted into mice and the response to theimplants is assessed. An exemplary bone portion 10 with a defect D isshown in FIG. 13.

Porcine bone implants are sterilely prepared and surrounding attachedsoft tissues surgically removed. The bone specimens are washed for atleast five minutes with an alcohol, such as ethanol or isopropanol, toremove synovial fluid and lipid soluble contaminants.

The bone specimens are frozen at a temperature ranging from about −35°C. to about −90° C., and preferably at a temperature up to about −70°C., to disrupt, that, is to kill, the specimens' bone cells.

Each bone specimen is cut into two portions. The first bone portion isimmersed in a buffer solution containing α-galactosidase at apredetermined concentration. The first bone portion is allowed toincubate in the buffer solution for a predetermined time period at apredetermined temperature. The second bone portion is incubated undersimilar conditions as the first bone portion in a buffer solution in theabsence of α-galactosidase and serves as the control.

At the end of the incubation, the bone portions are washed underconditions which allow the enzyme to diffuse out. Assays are performedto confirm the complete removal of the α-gal epitopes.

The α-galactosidase first bone portions disclosed above are thenimpregnated with demineralized bone matrix gel containing osteoinductivefactor prepared according to methods known in the prior art, such as,for example, John E. Feighan, et al., Induction of Bone by aDemineralized Bone Matrix Gel: A Study in a Rat Femoral Defect Model, 13Journal of Orthopaedic Research 881-889 (1995).

The bone samples are implanted in subcutaneous tissues of mice undergeneral inhalation anesthesia following known surgical procedures. Boneis implanted in subcutaneous tissues to evaluate the osteoinductiveproperties of bone. Any bone formed is evidence of osteoinductiveproperties. Osteoconductive properties of bone xenograft are evaluatedafter the xenograft is implanted using bone defective models such as thelong bone drill hole model. The implantation procedure is performedunder sterile surgical technique, and the wounds are closed with 3-0vicryl or a suitable equivalent known to those of ordinary skill in theart. FIG. 14 shows the bone portion 10 with the xenograft X (showncrosshatched) in place at the defect D. The animals are permittedunrestricted cage activity and monitored for any sign of discomfort,swelling, infection, or rejection. Blood samples (e.g., 2 ml) are drawnperiodically (e.g., every two weeks) for monitoring of antibodies.

The occurrence of an immune response against the xenograft is assessedby determining anti-Gal and non-anti-Gal anti-bone xenograft antibodies(i.e., antibodies binding to antigens other than the α-gal epitopes) inserum samples from the transplanted mice. Blood samples are drawn fromthe transplanted mice on the day of implant surgery and at periodic(e.g., two week) intervals post-transplantation. The blood samples arecentrifuged and the serum samples are frozen and evaluated for theanti-Gal and other non-anti-Gal anti-bone xenograft antibody activity.

Anti-Gal activity is determined in the serum samples in ELISA withα-gal-BSA as solid phase antigen, according to methods known in theprior art, such as, for example, the methods described in Galili et al.,Porcine and bovine cartilage transplants in cynomolgus monkey. II.Changes in anti-Gal response during chronic rejection, 63Transplantation 645-651 (1997).

Assays are conducted to determine whether α-galactosidase treatedxenografts induce the formation of anti-bone xenograft antibodies. Formeasuring anti-bone xenograft antibody activity, an ELISA assay isperformed according to methods known in the prior art, such as, forexample, the methods described in K. R. Stone et al., Porcine and bovinecartilage transplants in cynomolgus monkey: I. A modelfor chronicxenograft rejection, 63 Transplantation 640-645 (1997).

The bone xenografts are optionally explanted at one to two monthspost-transplantation, sectioned and stained for histological evaluationof inflammatory infiltrates. Post-transplantation changes in anti-Galand other anti-bone xenograft antibody activities are correlated withthe inflammatory histologic characteristics (i.e., granulocytes ormononuclear cell infiltrates) within the explanted bone, one to twomonths post-transplantation, using methods known in the art, as, forexample, the methods described in K. R. Stone et al., Porcine and bovinecartilage transplants in cynomolgus monkey: I. A modelfor chronicxenograft rejection, 63 Transplantation 640-645 (1997).

Where the bone xenograft is explanted, the bone xenograft is asepticallyharvested, using anesthetic procedure, removal of the implant andclosure of the soft tissue. Tissue is harvested for possible immunologictesting if the gross and histopathologic evaluation of the transplantsindicate good performance of the transplanted bone. The xenograftsamples are collected, processed, and examined microscopically. Aportion of the implant and surrounding tissue is frozen in an embeddingmedium for frozen tissue specimens in embedding molds forimmunohistochemistry evaluation according to the methods known in theprior art. “TISSUE-TEKT” O.C.T. compound which includes 10.24% w/wpolyvinyl alcohol, 4.26% w/w polyethylene glycol, and 86.60% w/wnonreactive ingredients, and is manufactured by Sakura FinTek, Torrence,Calif., is a non-limiting example of a possible embedding medium for usewith the present invention. Other embedding mediums known to those ofordinary skill in the art may also be used. The remaining implant andsurrounding tissue is collected in 10% neutral buffered formalin forhistopathologic examination.

EXAMPLE 6

Assessment Of Primate Response To Implanted Bone Xenografts Treated Withα-Galactosidase and Demineralized Bone Matrix Gel ContainingOsteoinductive Factor

In this example, porcine bone implants are treated with α-galactosidaseand demineralized bone matrix gel containing osteoinductive factor, theimplants are transplanted into cynomolgus monkeys, and the primateresponse to the bone implants is assessed, as described in Example 5.After the bone xenografts are explanted and tissue is harvested forpossible immunologic testing, the animals are allowed to recover and aremonitored closely until the incisions have healed and the gait of theanimals is normal.

EXAMPLE 7

Assessment Of Response In Mice To Implanted Bone Xenografts Treated Withα-Galactosidase, Demineralized Bone Matrix Gel Containing OsteoinductiveFactor, Fucosyl and Fucosyltransferase

In this example, porcine bone implants are treated with α-galactosidaseto eliminate α-gal epitopes, as described in Example 5. The implants arefurther treated with fucosyl and fucosyl transferase to cap carbohydratechains with fucosyl. Fucosyltransferase facilitates the transfer offucosyl to the xenograft. The fucosyl links to and thus caps thecarbohydrate chains. Capping with fucosyl interferes with the ability ofthe subject's immune system to recognize the xenograft as foreign. Theimplants are transplanted into mice, and the response to the boneimplants is assessed.

Porcine bone implants are prepared as described in Example 1 includingthe α-galactosidase treatment. Prior to implantation into the mice,however, the implants are further treated with a predetermined amount offucosyl and fucosyltransferase, at specified concentrations for apredetermined time and at a predetermined temperature, to capcarbohydrate chains with fucosyl. For example, the samples are immersedin a buffer solution at predetermined concentrations of fucosyl andfucosyl transferase. The samples are incubated for a predetermined timeperiod at a predetermined temperature.

Other molecules, such as n-acetyl glucosamine in combination with thecorresponding glycosyltransferase, can also be used for capping thecarbohydrate chains of the implants.

Subsequently, the samples are washed to remove the enzyme and implantedinto the mice, and the occurrence of an immune response against thexenograft is assessed as described above in Example 5.

EXAMPLE 8

Assessment Of Primate Response To Implanted Bone Xenografts Treated Withα-Galactosidase, Demineralized Bone Matrix Gel Containing OsteoinductiveFactor; Fucosyl and Fucosyltransferase

In this example, porcine bone implants are treated with α-galactosidase,demineralized bone matrix gel containing osteoinductive factor, fucosyland fucosyltransferase; the implants are transplanted into cynomolgusmonkeys and the primate response to the bone implants is assessed, asdescribed above in Examples 5, 6 and 7.

EXAMPLE 9

Assessment Of Response In Mice To Implanted Bone Xenografts Treated WithAlcohol and Freeze/Thaw Cycling

In this example, porcine bone implants are treated with alcohol andfreeze/thaw cycling prior to their transplantation in mice and theresponse of the mice to the implants is assessed.

Porcine bone implants are sterilely prepared and surrounding attachedsoft tissues surgically removed. The resultant xenograft is washed inabout ten volumes of sterile cold water to remove residual bloodproteins and water soluble materials. The xenograft is then immersed inalcohol at room temperature for about five minutes, to sterilize thebone and to remove non-collagenous materials.

After the alcohol washing, the xenograft is treated by again placing thexenograft in an alcohol solution of 70% isopropanol at room temperaturefor at least five minutes.

Following the alcohol treatment step, the bone implants are placed in afreezer until the xenograft is completely frozen, i.e., no interior warmspots remain which contain unfrozen tissue. Each bone implant is thenthawed by immersion in an isotonic saline bath at room temperature(about 25° C.) for about ten minutes.

It should be understood that the bone implants alternatively can besubjected to the above-described freeze/thaw cycling treatment prior tothe alcohol treatment step.

The implants are then implanted in the mice and the occurrence of animmune responses against the xenograft is assessed as described above inExample 5.

EXAMPLE 10

Assessment Of Primate Response To Implanted Bone Xenografts Treated WithAlcohol and Freeze/Thaw Cycling

In this example, porcine bone implants are treated with alcohol andfreeze/thaw cycling prior to their transplantation in cynomolgus monkeysand the primate response to the implants is assessed as described abovein Examples 5-10.

EXAMPLE 11

Assessment Of Response In Primates To Implanted Soft Tissue And BoneXenografts Treated With α-Galactosidase And Glutaraldehyde

In this example, porcine meniscal, articular cartilage, ligament,peritoneum heart valve and bone implants are treated with alcohol andfreeze/thaw cycling, as described above in examples 1-10. They arefurther treated with between 0.01-0.10% glutaraldehyde, subsequentlytreated with α-galactosidase, and the primate response to the implants,is assessed as described above in examples 1-10.

Those of skill in the art will recognize that the invention may beembodied in other specific forms without departing from the spirit oressential characteristics thereof. The presently described embodimentsare therefore to be considered in all respects as illustrative and notrestrictive, the scope of the invention being indicated by the appendedclaims rather than by the foregoing description, and all variations ofthe invention which are encompassed within the meaning and range ofequivalency of the claims are therefor intended to be embraced therein.

What is claimed is:
 1. A method of preparing a xenograft formed of asoft or bone tissue for implantation into a human, which comprises a.removing at least a portion of the soft or bone tissue from a non-humananimal to provide a xenograft; b. washing the xenograft in water andalcohol; c. subjecting the xenograft to a cellular disruption treatment;d. exposing the xenograft to an aldehyde in an amount ranging from about0.01% to about 0.10%; and e. digesting the xenograft with a glycosidaseto remove substantially a plurality of first surface carbohydratemoieties from the xenograft whereby the xenograft is substantiallynon-immunogenic and has substantially the same mechanical properties asthe native soft or bone tissue.
 2. A method according to claim 1,wherein the step of exposing the xenograft to an aldehyde comprisesexposing the aldehyde to an aldehyde selected from the group consistingof glutaraldehyde, formaldehyde, and adipic dialdehyde.
 3. A methodaccording to claim 1 further comprising the step of after step c,piercing the xenograft.
 4. A method according to claim 1 furthercomprising the step of after step c, treating the xenograft with asecond enzyme.
 5. A method according to claim 4, wherein the secondenzyme is selected from the group consisting of ficin and trypsin.
 6. Amethod according to claim 1 further comprising the step of after step c,treating the xenograft with one or more agents selected from the groupconsisting of anticalcification agents, antithrombotic agents,antibiotics, and growth factors.
 7. A method according to claim 1further comprising the step of after step c, sterilizing the xenograft.8. A method according to claim 7, wherein the sterilizing step comprisessterilizing the xenograft with one or more agents selected from thegroup consisting of ethylene oxide and propylene oxide.
 9. A methodaccording to claim 1 further comprising the step of: after step c,treating the xenograft with polyethylene glycol.
 10. A method accordingto claim 1 further comprising the step of after step c, exposing thexenograft to one or more agents selected from the group consisting ofaliphatic diamine compounds and aromatic diamine compounds.
 11. A methodaccording to claim 1, wherein the cellular disruption treatmentcomprises freeze/thaw cycling.
 12. A method according to claim 1,wherein the cellular disruption treatment comprises exposure to gammaradiation.
 13. A method according to claim 1, wherein the glycosidase isa galactosidase.
 14. A method according to claim 13, wherein thegalactosidase is an α-galactosidase.
 15. A method according to claim 1further comprising the step of after step e, treating a plurality ofsecond surface carbohydrate moieties on the xenograft with a pluralityof capping molecules to cap at least a portion of the second surfacecarbohydrate moieties.
 16. A method according to claim 15, wherein atleast a portion of the capping molecules are a plurality of fucosylmolecules.
 17. A method according to claim 15, wherein at least aportion of the capping molecules are a plurality of n-acetyl glucosaminemolecules.
 18. A method according to claim 1 further comprising the stepof after step d, removing substantially a plurality of proteoglycansfrom the xenograft, wherein the xenograft is a soft tissue.
 19. A methodaccording to claim 18, wherein the removing step comprises digesting thexenograft with one or more proteoglycan-depleting factors selected fromthe group consisting of chondroitinase ABC, hyaluronidase, chondroitinAC II lyase, keratanase, trypsin and fibronectin fragments.
 20. A methodaccording to claim 1, wherein the removing step comprises removing theportion of a medial or lateral meniscus having a superior principalsurface and an inferior principal surface, each of the principalsurfaces having an outer portion being joined by an outer lateralsurface, and each of the principal surfaces having an inner portionbeing joined by an inner lateral surface.
 21. A method according toclaim 1, wherein the removing step comprises removing the portion of aligament.
 22. A method according to claim 21, wherein the removing stepcomprises removing with the portion a first block of bone attached to afirst end of the portion.
 23. A method according to claim 22, whereinthe removing step comprises removing with the portion a second block ofbone affixed to a second end of the portion opposite the first end. 24.A method according to claim 1, wherein the removing step comprisesremoving the portion of an articular cartilage.
 25. A method accordingto claim 24, wherein the removing step comprises removing with theportion a layer of subchondral bone.
 26. An article of manufacturecomprising a substantially non-immunogenic aldehyde-treated,glycosidase-treated xenograft formed of a soft or bone tissue forimplantation into a human, produced by the process of a. removing atleast a portion of the soft tissue, hard tissue or heart valve from anon-human animal to provide a xenograft; b. washing the xenograft inwater and alcohol; c. subjecting the xenograft to a cellular disruptiontreatment; d. exposing the xenograft to an aldehyde in an amount rangingfrom about 0.01% to about 0.10%; and e. digesting the xenograft with aglycosidase to remove substantially a plurality of first surfacecarbohydrate moieties from the xenograft whereby the xenograft issubstantially non-immunogenic and has substantially the same mechanicalproperties as the native soft or bone tissue.
 27. An article ofmanufacture according to claim 26, wherein the aldehyde is selected fromthe group consisting of glutaraldehyde, formaldehyde, and adipicdialdehyde.
 28. An article of manufacture according to claim 26, whereinthe xenograft has a plurality of punctures for increasing permeabilityto agents and enzymes.
 29. An article of manufacture according to claim26 further comprising one or more agents selected from the groupconsisting of anticalcification agents, antithrombotic agents,antibiotics, and growth factors.
 30. An article of manufacture accordingto claim 26, wherein the xenograft is sterilized.
 31. An article ofmanufacture according to claim 26, wherein the xenograft is apolyethylene glycol-treated xenograft.
 32. An article of manufactureaccording to claim 26, further comprising an agent selected from thegroup consisting of aliphatic diamine compounds and aromatic diaminecompounds.
 33. An article of manufacture according to claim 26, whereinthe glycosidase-treated xenograft is a galactosidase-treated xenograft.34. An article of manufacture according to claim 33, wherein thegalactosidase-treated xenograft is an α-galactosidase-treated xenograft.35. An article of manufacture according to claim 26 further comprising aplurality of capping molecules capped on a plurality of second surfacecarbohydrate moieties on the xenograft.
 36. An article of manufactureaccording to claim 35, wherein the capping molecules comprise aplurality of fucosyl molecules.
 37. An article of manufacture accordingto claim 35, wherein the capping molecules comprise a plurality ofn-acetyl glucosamine molecules.
 38. An article of manufacture accordingto claim 26, wherein the xenograft is a thawed, frozen xenograft.
 39. Anarticle of manufacture according to claim 26, wherein the xenograft is agamma-irradiated xenograft.
 40. An article of manufacture according toclaim 26, wherein the xenograft is a proteoglycan-depletingfactor-treated xenograft, and wherein one or more of theproteoglycan-depleting factors is selected from the group consisting ofchondroitinase ABC, hyaluronidase, chondroitin AC II lyase, keratanase,trypsin, and fibronectin fragments.
 41. An article of manufactureaccording to claim 26, wherein the portion is of a medial or lateralmeniscus having a superior principal surface and an inferior principalsurface, each of the principal surfaces having an outer portion beingjoined by an outer lateral surface, and each of the principal surfaceshaving an inner portion being joined by an inner lateral surface.
 42. Anarticle of manufacture according to claim 26, wherein the portion is ofa ligament.
 43. An article of manufacture according to claim 42, whereinthe portion of the ligament comprises a first block of bone attached toa first end of the portion.
 44. An article of manufacture according toclaim 43, wherein the portion of the ligament comprises a second blockof bone affixed to a second end of the portion opposite the first end.45. An article of manufacture according to claim 26, wherein the portionis of an articular cartilage.
 46. An article of manufacture according toclaim 45, wherein the portion of the articular cartilage comprises alayer of subchondral bone.
 47. A xenograft formed of a soft or bonetissue for implantation into a human, comprising: a portion of the softor bone tissue from a nonhuman animal, wherein the portion includes aplurality of extracellular components, a plurality of substantially onlydead cells, and a plurality of proteins within said extracellularcomponents, said protein s crosslinked with an aldehyde in an amountranging from about 0.01% to about 0.10%, the extracellular componentsand the dead cells having substantially no surface carbohydrate moietieswhich are susceptible to glycosidase digestion, and whereby the portionis substantially non-immunogenic and has substantially the samemechanical properties as a corresponding portion of the native soft orbone tissue.
 48. A xenograft according to claim 47, wherein the aldehydeis in an amount ranging from about 0.01% to about 0.05% crosslinking theproteins.
 49. A xenograft according to claim 47, wherein the portion hascapping molecules linked to at least a portion of a plurality of surfacecarbohydrate moieties on the xenograft, and whereby the xenograft issubstantially non-immunogenic.
 50. A xenograft according to claim 49,wherein at least a portion of the capping molecules are a plurality offucosyl molecules.
 51. A xenograft according to claim 49, wherein atleast a portion of the capping molecules are a plurality of n-acetylglucosamine molecules.
 52. A xenograft according to claim 47, whereinthe portion is of a medial or lateral meniscus having a superiorprincipal surface and an inferior principal surface, each of theprincipal surfaces having an outer portion being joined by an outerlateral surface, and each of the principal surfaces having an innerportion being joined by an inner lateral surface.
 53. A xenograftaccording to claim 47, wherein the portion is of a ligament.
 54. Axenograft according to claim 53, wherein the portion of the ligamentcomprises a first block of bone attached to a first end of the portion.55. A xenograft according to claim 54, wherein the portion of theligament comprises a second block of bone affixed to a second end of theportion opposite the first end.
 56. A xenograft according to claim 47,wherein the portion is of an articular cartilage.
 57. A xenograftaccording to claim 56, wherein the portion of the articular cartilagecomprises a layer of subchondral bone.
 58. A method according to claim1, wherein the step of exposing the xenograft to an aldehyde comprisesexposing the xenograft to the aldehyde in an amount ranging from about0.01% to about 0.05%.
 59. An article of manufacture according to claim26, wherein the xenograft is produced by exposing the xenograft to thealdehyde in an amount ranging from about 0.01% to about 0.05%.